Recombinant Adenoviruses (Ad) show great promise as vectors for the treatment of disease by gene therapy. Somatic gene therapy is defined as the treatment of inherited or acquired diseases by the introduction and expression of genetic information in somatic cells. Candidate disorders for somatic gene therapy include inherited metabolic disorders as well as acquired diseases including several types of cancer. Because of their intrinsic ability to enter cells carrying along their own genetic material, viruses are widely used as gene delivery vectors for living organisms. Advantages of employing Ad as a gene therapy vector are many: the genetic organization and functions of many gene products have been characterized, the virus infects a wide host cell range, the virus is capable of productive infection of resting cells, the genome is easily manipulated, and recombinant viruses can be grown to high titer in the laboratory. Finally, Ad infection is relatively benign and has not been associated with any human malignancy. It has been shown in animal studies and human clinical trials however, that Ad invokes an inflammatory response soon after infection, followed by a cytotoxic T cell response directed against virus-infected cells. Innate host defenses, such as recruitment of macrophages and activation of complement and natural killer cells, are thought to play a substantial role in the clearance of an Ad infection iii vivo (Worgall, Leopold et al. 1997; Worgall, Singh et al. 1999). This clearance of virus-infected cells ultimately leads to cessation of foreign gene expression. A number of classes of Ad vectors exist, each eliciting the immune response to varying degrees.
Viruses rendered replication-defective by the deletion of one or more early genes have demonstrated their potential usefulness in animal models of cancer for example (Addison, Braciak et al. 1995; Addison, Gauldie et al. 1995). In such first generation vectors, the E1 and/or E3 gene cassettes are removed, allowing the introduction of up to 6.5 kb of transgene (Graham, Smiley et al. 1977) under the control of a heterologous promoter. Removal of the E1 region results in impaired transcription of the E2 genes and, consequently, an impairment of viral DNA replication and production of viral proteins, and thereby reduces the host immune response to viral proteins. The defective E1 viruses are propagated in an E1-complementing cell line, such as 293 cells, that provide the E1 gene products in trans. Although some first generation Ad vectors have shown promise for certain applications, such as cancer therapy, the expression of the transgene in vivo has been shown to be transient, and an overwhelming immune response is mounted by the host. Deletion of the E3 coding region allows for larger transgene inserts, but further renders the cells susceptible to apoptosis, as a result of the elimination of the E3 gene-mediated defenses against host responses (Poller, Schneider-Rasp et al. 1996).
Second and third generation vectors lack the E2 genes, in addition to the E1 and E3 genes (Lusky, Christ et al. 1998; O'Neal, Zhou et al. 1998). Even with the E2 deletions, however, the host immune response is still a daunting impediment to achieving persistent transgene expression. The generation of replication-competent Ad (RCA) through recombination between Ad sequences within the cell line and the recombinant vector has prevented the production of pure stocks of all of the early generation vectors.
Improvements made on early generation vectors have culminated in the development of helper-dependent (HD) or gutted vectors deleted of most or all Ad coding sequences. Gutted vectors allow for nearly 36 kb of transgene sequences as well as a reduced host immune response (Kochanek, Clemens et al. 1996; Parks, Chen et al. 1996). These vectors show great potential as gene transfer vectors for gene therapy as long-term expression of therapeutic genes has been observed in mice and monkeys (Chen, Mack et al. 1997; Morral, Parks et al. 1998; Morsy, Gu et al. 1998; Schiedner, Morral et al. 1998; Morral, O'Neal et al. 1999). The production of such vectors in tissue culture relies upon a complementing helper virus to provide the proteins in trans that are required for growth and assembly of the gutted vector. A significant barrier to wider application of gutted vectors, however, is the production of pure gutted vector stocks. Although the gutted vector lacks virtually all Ad sequences, except those cis-acting elements required for DNA replication and packaging, the use of these vectors has been limited by high levels of contaminating helper virus. Contamination with high levels of helper virus is unacceptable in a gene therapy application. One approach to limit contamination with helper virus is to alter the packaging sequences of the two vectors to eliminate sequence overlap (Sandig, Youil et al. 2000). A second approach to limit contamination with helper virus is biological selection based on Cre recombinase-mediated excision of the helper genome's packaging signal via loxP sites during gutted vector production (Parks, Chen et al. 1996; Hardy, Kitamura et al. 1997). In this Cre-lox system, the helper virus contains two loxP recognition sequences flanking the packaging domain. To produce the gutted vector, cells that express the Cre-recombinase of bacteriophage P1 are coinfected with the helper virus and gutted vector. In these production cells, the packaging domain of the helper virus is excised with high efficiency so that packaging of the helper virus is greatly reduced and the majority of packaged genomes are gutted virus genomes. This method of production reduces the contamination of gutted vector with helper virus to ≦1% after purification of virus particles by CsCl centrifugation. Although this system has resulted in higher titers of gutted vector preparations with low helper virus contamination compared to other systems, the Cre-mediated excision is not 100% effective and all groups report some level of contamination.
The AdS packaging domain consists of at least seven cis-acting sequences termed A repeats I-VII. It is thought that these cis-acting sequences are binding sites for an unidentified trans-acting protein(s), and that this interaction is required to mediate DNA packaging. In accordance with the present invention, binding sites for the E. coli Lac repressor protein have been inserted within the packaging domain of the helper virus and between the critical A repeats. One domain of the repressor protein binds to an operator sequence that shares some homology with the Ad packaging domain, while another domain binds the inducer isopropyl-β-D-thiogalactopyranoside (IPTG). In place of the Ad E1 genes, a CMV promoter-driven lacI expression cassette has been inserted. In the presence of IPTG, the virally expressed Lac repressor is not bound to the packaging domain and DNA packaging of the helper virus genome ensues. In the absence of IPTG, however, Lac repressor protein binds to its sites embedded within the packaging domain of the helper virus genome. This binding by Lac repressor protein precludes the natural packaging factor(s) from binding and therefore DNA packaging of the helper virus genome is suppressed. The subject Ad helper virus is capable of specifically suppressing its own DNA packaging while providing all of the proteins required in trans to replicate and package a gutted Ad genome. This method of regulation, used alone or in combination with the Cre-lox system, results in a helper Ad capable of generating high-titer gutted Ad vector stocks substantially free of helper virus contamination.